Preservation of aqueous systems with 2-chloro-3-oxobutyramide derivatives

ABSTRACT

There is disclosed methods and compositions for inhibiting or preventing the growth of microorganisms in aqueous systems wherein the growth is inhibited or prevented by the presence of an effective amount of a 2-chloro-3-oxobutyramide derivative.

This is a division of application Ser. No. 934,309 filed Aug. 17, 1978.

THE INVENTION

A number of aqueous systems are susceptible to antimicrobial growth.Among these are cosmetics latex paints, polymer emulsions, industrialoil in water emulsions, adhesives, water used in industrial coolingtowers, white water in the paper mills and the like. The growth ofbacteria and fungi in such systems can be a serious problem if notproperly controlled. For example, industrial aqueous systems aresusceptible to slime formation which, if unchecked, can cause severemaintainance and production problems. Similarly, consumer products suchas cosmetics can be damaged by the growth of bacteria, fungi or algae.

There is, consequently, a continuing effort to provide effective andeconomical antimicrobial agents which protect these systems. The findingof this invention is that compositions and methods utilizing2-chloro-3-oxobutyramide derivatives provide effective control of suchantimicrobial growth. The compounds of this invention have been foundeffective against a broad spectrum of microorganisms including grampositive bacteria, gram negative bacteria and molds. The breadth of suchactivities is illustrated in the examples.

The 2-chloro-3-oxobutyramide derivatives of this invention can berepresented by the formula: ##STR1## wherein R₁ is hydrogen or methyland R₂ is selected from the group consisting of hydrogen, methyl,alkanoyl or carboalkoxy.

Two of these compounds have been reported in the chemical literature.The 2-chloro-3-oxobutyramide is reported in Chemical Abstracts 42:p2615aand the 2-chloro-N,N-dimethyl-3-oxobutyramide (also known asα-chloro-N,N-dimethylacetoacetamide) is reported in Chemical Abstracts52:2067c. Neither reference discloses or suggests the effectiveness ofthese compounds in preventing antimicrobial growth.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Formula I represents the compounds of this invention. While theN,N-disubstituted analogs such as 2-chloro-N,N-dimethyl-3-oxobutyramideshow a high level of activity, those in which R₁ is hydrogen aresomewhat more active and are preferred.

Those compounds of formula I wherein R₂ is acyl or carboalkoxy arenovel. Of these, the lower analogs wherein R₂ is acetyl, propionyl,butyroyl, carbomethoxy, carbethoxy, carbopropoxy and carbobutoxy arepreferred. Especially preferred are N-acetyl-2-chloro-3-oxobutyramide,N-carbomethoxy-2-chloro-3-oxobutyramide andN-carbethoxy-2-chloro-3-oxobutyramide.

The compounds of this invention may be added to the aqueous systems orformulations undiluted or dissolved in organic solvents such asalcohols, acetone, dimethylformamide and the like. They may be addedalone or in combination with other biocides and/or functional compoundssuch as antioxidants, anticorrosive agents, surfactants, etc.

Concentrations from about 0.005% to above 0.3% are effective. Use oflarger concentrations, while feasible, is recommended only for unusualapplications. It is preferred to use concentrations from about 0.05% toabout 0.15%.

The compounds of this invention can be used as preservatives for oil inwater emulsions. A number of oil water emulsions are used in industry,for example in the high speed metal working and textile industries, fortheir cooling, lubricating, antistatic and anticorrosive properties.Unless adequately protected by an effective preservative, such systemsare susceptible to bacterial decomposition producing obnoxious odors andpotential health hazards. [Detailed descriptions of these systems, theirmicrobiological problems and difficulties in their preservation can befound in Bennet, E. O., Soap Chem. Specialties, 32, 46 (1956) andFabian, F. W. & Pivnick, H., Applied Microbiology, 1, (1953).]

In practicing the invention, the compounds may be added by directlydissolving them in the concentrated oil and then mixing with water toform the water oil emulsion, or they may be added to the final emulsioneither undiluted or dissolved in a solvent such as dimethylformamide,alcohol, acetone, etc. Similar methods known in the art for addingpreservatives to water and oil emulsions may also be used.

There can be used as little as about 0.005%. Although amounts greaterthen 0.3% are operable, they are recommended only for unusualapplications. It is preferred to use amounts in the range of from about0.01% to about 0.2%, with amounts in the range of about 0.02% to 0.10%being especially preferred.

The 2-chloro-3-oxobutyramide derivatives are also effective as cosmeticpreservatives [Problems encountered in the preservation of cosmetics aredescribed by Dunnigan, A. P., Drug and Cosmetic Industries, 103, 43,(1968)].

The compounds may be added to the finished cosmetic product directly ordissolved in suitable solvents such as alcohol, acetone, dimethylformamide and the like. Alternatively the compounds may be dissolved inthe oils or other raw materials used in the formula and then formulatedin the final product.

In cosmetic preparations, concentrations as low as 0.01% are found to beoperable. Concentrations greater than 0.30%, while operable, arerecommended only for unusual applications. Concentrations in the rangeof from about 0.02% to about 0.2% are preferred with concentrations ofabout 0.05% to 0.10% being especially preferred.

The 2-chloro-3-oxobutyramides derivatives are effective as slimicides.For example it can be used to protect so-called white water systemsutilized in paper manufacture from the formation of slimes and the likewhich are known to affect these systems. Concentrations as low as 0.005%are found to be operable. Concentrations greater than 0.20%, whileoperable, are recommended only for unusual applications. Concentrationsin the range of from about 0.007% to about 0.15% are preferred withconcentrations of about 0.01% to about 0.10% being especially preferred.

While the compounds are effective when added directly, it is preferredto add them dissolved in a suitable solvent such as diethylene glycol,dipropylene glycol or polyethylene glycol and the like. Other methodsknown in the art for adding preservatives to such aqueous systems mayalso be used.

ILLUSTRATION OF PREFERRED EMBODIMENTS

A number of examples are provided herein to illustrate the preferredembodiments of this invention. The examples provided are included forthe sole purpose of illustrating the preferred embodiments and shouldnot be construed as limiting. They are intended to embrace anyequivalents or obvious extensions which are known or should be known toa person skilled in the art.

EXAMPLE I General Antimicrobial Activity

General antibacterial and antifungal activity was evaluated by a 5-foldserial dilution test in agar. In this test, compounds were prepared as6% solutions in dimethylformamide or ethanol. The 6% solution was then5-fold serially diluted in test tubes to give the desired concentrationswhen mixed with agar and poured into sterile Petri dishes. Tryptoneglucose extract agar was used for the bacterial testing; mildew glucoseagar for the fungal testing. The bacterial plates were spot inoculatedwith 24-hour nutrient broth cultures and incubated at 37° C. for 48hours. The fungal plates were spot inoculated with spore suspensions andincubated at 28° C. for seven days. At the end of the incubationperiods, all plates were examined for growth. The minimum inhibitoryconcentration (MIC) for each organism is expressed in Table I. In theranges presented, growth is observed only at the lower concentration.

The activity is presented in Tabular Form (Table I) using the followingkey.

    ______________________________________                                        Bacteria          Fungi                                                       ______________________________________                                        B.sub.1 Staphylococcus aureus                                                                   F.sub.1 Aspergillus niger                                   B.sub.2 Escherichia coli                                                                        F.sub.2 Aspergillus oryzae                                  B.sub.3 Pseudomonas aeruginosa                                                                  F.sub.3 Penicillium piscarium                               B.sub.4 Proteus vulgaris                                                                        F.sub.4 Aureobasidium pullulans                             B.sub.5 Bacillus subtilis                                                     ______________________________________                                        ACTIVITY RANGE*                                                               ______________________________________                                        0 = >1920 mcg/ml  5 = 0.6-3.1 mcg/ml.                                         1 = 384-1920 mcg/ml.                                                                            6 = 0.12-0.6 mcg/ml.                                        2 = 76.8-384 mcg/ml.                                                                            7 = 0.03-0.12 mcg/ml.                                       3 = 15.3-76.8 mcg/ml.                                                                           8 = <0.30 mcg/ml.                                           4 = 3.1-15.3 mcg/ml.                                                          ______________________________________                                         *In the ranges presented, growth is observed only in the lower                concentrations.                                                          

                  TABLE I                                                         ______________________________________                                         ##STR2##                                                                                BACTERIA    FUNGI                                                  R.sub.1                                                                              R.sub.2   B.sub.1                                                                             B.sub.2                                                                           B.sub.3                                                                           B.sub.4                                                                           B.sub.5                                                                           F.sub.1                                                                           F.sub.2                                                                           F.sub.3                                                                           F.sub.4                    ______________________________________                                        H      H         3     3   3   3   3   3   3   3   3                          H      COCH.sub.3                                                                              2     3   3   2   2   2   2   2   2                          H      COOC.sub.2 H.sub.3                                                                      2     2   2   2   2   2   2   2   2                          CH.sub.3                                                                             CH.sub.3  1     2   1   2   1   1   1   1   2                          ______________________________________                                    

EXAMPLE II Utility as a Cosmetic Preservative

The 2-Chloro-3-oxobutyramide derivatives are effective cosmeticpreservatives. Two-fold serial dilutions of 6% solutions of the2-haloacetoacetic acid derivatives in dimethylformamide were added to acosmetic lotion of the following formulation:

    ______________________________________                                        Stearic acid            1.4 g                                                 Arlacel 60 (sorbitan                                                          monostearate)           0.7 g                                                 Tween 20 Polyoxethylene                                                       (20) sorbitan                                                                 monostearate            1.6 g                                                 Distilled water         94.0 g                                                ______________________________________                                    

The lotions were inoculated with both Pseudomonas aeruginosa andAspergillus niger and incubated at 28° C. At weekly intervals, thelotions were examined for microorganisms by conventional streak-platemethods or by macroscopic observation. The lotions were thenreinoculated with the test organisms and reincubated. Table II shows theminimum inhibitory concentration that was effective in preventingmicrobial growth for the four week period.

                  TABLE II                                                        ______________________________________                                         ##STR3##                                                                                 Pseudomonas aeruginosa                                                                        Aspergillus niger                                 Compound    Week            Week                                              R.sub.1                                                                             R.sub.2   1      2    3    4    1   2   3   4                           ______________________________________                                        H     H         5      5    4    4    5   5   4   2                           H     COCH.sub.3                                                                              5      5    4    4    5   4   3   2                           H     COOC.sub.2 H.sub.5                                                                      5      4    4    4    4   3   3   3                           CH.sub.3                                                                            CH.sub.3  2      2    2    2    1   1   1   1                           ______________________________________                                         Key:                                                                          0 = >2000 mcg/gm                                                              1 = 1000-2000 mcg/gm                                                          2 = 500-1000 mcg/gm                                                           3 = 250-500 mcg/gm                                                            4 = 125-250 mcg/gm                                                            5 = 63-125 mcg/gm                                                             6 = 32-62.5 mcg/gm                                                            7 = 16-31.3 mcg/gm                                                            8 = 8-15.6 mcg/gm                                                             9 = >8 mcg/gm                                                            

EXAMPLE III

The utility of the 2-chloro-3-oxobutyramide derivatives in water and oilemulsions is illustrated below using a commercially available cuttingoil.

In running these tests, two-fold serial dilutions of 6% solutions of thecompound in dimethylformamide was added to 3.3% cutting oil emulsions.The emulsions were prepared by diluting with water a commerciallyavailable cutting oil concentrate. The emulsions were inoculated with aculture of Pseudomonas aeruginosa and incubated at 28° C. on a rotaryshaker. At weekly intervals, the emulsions were examined for bacteria byconventional streak-plate methods. The emulsions were then reinoculatedwith Pseudomonas aeruginosa and reincubated.

                  TABLE III                                                       ______________________________________                                         ##STR4##    Minimum Inhibitory Concentration Range   (Micrograms/millilit                 er)                                                                           Psuedomonas aeruginosa                                           Compound     Week                                                             R.sub.1                                                                             R.sub.2    1        2      3      4                                     ______________________________________                                        H     H          6        3      3      3                                     H     COCH.sub.3 4        3      2      2                                     H     COOC.sub.2 H.sub.5                                                                       4        3      3      2                                     CH.sub.3                                                                            CH.sub.3   2        1      1      1                                     ______________________________________                                    

The highest level of the chemical inhibiting growth and the lowest levelat which growth occurs indicate the activity range for that compound.

    ______________________________________                                        0 = >500 mcg/ml     4 = 32-63 mcg/ml                                          1 - 250-500 mcg/ml  5 = 16-32 mcg/ml                                          2 = 125-500 mcg/ml  6 = 8-16 mcg/ml                                           3 = 63-125 mcg/ml   7 = 4-8 mcg/ml                                                                8 = 2-4 mcg/ml                                            ______________________________________                                    

EXAMPLE IV

The utility of the 2-Chloro-3-oxobutyramide derivatives as a slimicidefor pulp and paper mill water systems was demonstrated by the followingstudy.

Various quantities of a 6% solution of this compound indimethylformamide were incorporated into 24 ml of a test substratecomposed as follows:

84.4 g Whatman No. 2 powdered cellulose

2.6 g Sodium nitrate

1.0 g Calcium sulfate

6.5 g Maltose

1.0 g Nutrient Broth, Difco

10.0 ml Mersize Rm 70R (Monsanto)

2.5 ml 2% Alum

900 ml Distilled water

The samples were inoculated with four different organisms and incubatedat 28° C. At weekly intervals the samples were examined for the presenceof microbial growth and reinoculated during a total incubation period offour weeks. The results are tabulated in Table IV.

                  TABLE IV                                                        ______________________________________                                        Effectiveness of 2-Chloro-3-oxobutyramides Derivatives                        as Slimicides (mcg/ml)                                                        ______________________________________                                        A. 2-Chloro-3-oxobutyramide                                                            Weeks*                                                               Organism  1         2         3       4                                       ______________________________________                                        P. aeruginosa                                                                           62.5      62.5      62.5    62.5                                    E. aerogenes                                                                            62.5      62.5      62.5-125                                                                              62.5-125                                A. niger  62.5      62.5      62.5    62.5                                    P. piscarium                                                                            62.5      62.5-125   125-250                                                                               125-250                                ______________________________________                                        B. N-Acetyl-2-Chloro-3-oxobutyramide                                                  Weeks*                                                                Organism  1         2         3       4                                       ______________________________________                                        P. aeruginosa                                                                           62.5      62.5      62.5    62.5                                    E. aerogenes                                                                            62.5-125   125-250   250-500                                                                               250-500                                A. niger  62.5-125  62.5-125  62.5-125                                                                              62.5-125                                P. piscarium                                                                            62.5      62.5-125  62.5-125                                                                              62.5-125                                ______________________________________                                         *Data is given in mcg/ml. In a given range growth occurs at the lower         concentration and no growth occurs at the higher concentration. The lowes     concentration tested was 62.5 mcg/ml and that value given above indicates     that no growth was observed at the minimum concentration.                

EXAMPLE V

The following illustrates methods by which the compounds used inExamples I-IV may be prepared.

2-Chloro-3-oxobutyramide

(a) From α-chloro-β-aminocrotononitrile: The nitrile (5 g) was added toa solution of sulfuric acid (5 g) in water (10 ml) and the solution keptfor two days. This resulted in partial hydrolysis toα-chloro-α-cyanoacetone which formed a separate layer. This wasextracted into ether, the ether evaporated, and the residue taken up inconc. HCl (10 ml) and left overnight. A precipitate of ammonium chloride(900 mg) was filtered out and washed with ether, the filtrate wasevaporated to a small volume and ether (50 ml) was added. This mixturewas dried with Na₂ SO₄, filtered, evaporated, the residue taken up inwarm CH₂ Cl₂, dried again and evaporated. The residue solidified onstanding, it was triturated with a small volume of CH₂ Cl₂, filtered andwashed with CH₂ Cl₂ and then dried to give 1.54 g of colorless crystals.By concentrating the filtrate to ca. 2 ml a second crop of 0.63 g wasobtained (combined yield=37%).

(b) From α-chloro-α-cyanoacetone: A mixture of this material (80 g) inconc. HCl (100 ml) required cooling in ice to control the hydrolysisreaction. When the exotherm subsided, the solution was left overnightand worked up as in (a) to give 28.5 g (31%) of the amide in threecrops. An analytical sample prepared by vacuum sublimation had m.p.78°-85°, ir (CHCl₃) 3200-3500, 1730 (shoulder), 1700, 1630, and 1575cm⁻¹ ; nmr (CDCl₃) 2.11 and 2.40 (CH₃ of ketone and enol), 4.80 (CH ofketone), and 6.5 (broad, NH₂ and OH of enol); and mass spectrum m/e 43(100%) and 135 (M⁺).

Anal. Calcd for C₄ H₆ ClNO₂ : C, 35.44; H, 4.46; Cl, 26.15; N, 10.33.Found: C, 35.39; H, 4.50; Cl, 26.31; N, 10.36.

(c) From α-chloro-β-aminocrotonamide: A mixture ofα-chloro-β-aminocrotonamide (4 g), and conc. HCl (4 ml), in ether (4 ml)was stirred for 1 hr. The mixture was then diluted with CH₂ Cl₂ (20 ml),dried with Na₂ SO₄ and decanted. The solid was thoroughly washed severaltimes with ether and the combined organic layers evaporated. The wetresidue was taken up in ether, diluted with benzene and again evaporatedto give 3.8 g (94.3%) of pale yellow ketone, identified by ir, tlc andmp.

N-Acetyl-2-chloro-3-oxobutyramide

A solution of 2-chloro-3-oxobutyramide (1 g) in acetic anhydride (5 ml)containing acetyl chloride (1 ml) was heated in a 100° oil bath for 2hours. Excess reagent was evaporated under reduced pressure and theresidue was vacuum distilled in a kugelrohr apparatus. The distilledproduct crystallized to give a colorless solid with mp 54°-8°; ir(Nujol) 3200 and 1700 cm⁻¹ (broad); nmr (CDCl₂) 2.28 (CH₃), 2.47 (CH₃),5.53 (CH), and 9.7 ppm (NH), (a weaker set of singlets corresponding tothe enol appears at 2.23, 2.52, and 8.7 ppm); mass spectrum m/e 43(100%), 93, 135, and 177 (M⁺).

Anal. Calcd for C₆ H₈ ClNO₃ : C, 40.58; H, 4.54; Cl, 19.97; N, 7.89.Found: C, 40.83; H, 4.40; Cl, 20.08; N, 8.03.

N-Carboethoxy-2-chloroacetoacetamide

A mixture of diketene (25 g), urethane (20 g), and acetic acid (75 ml)was refluxed for 25 minutes. Evaporation of the acetic acid underreduced pressure gave a syrup which crystallized on scratching.Recrystallization from benzene gave 16.6 g (32.5%) ofN-carbethoxyacetoacetamide as colorless needles.

The N-carbethoxyacetoacetamide (12.7 g from above reaction) wasdissolved in methylene chloride (50 ml) and cooled in an ice bath.Sulfuryl chloride (10.0 g) was added dropwise to the reaction mixture.After refluxing for 45 minutes the solvent was evaporated under reducedpressure. The resulting colorless oil crystallized over night to give15.2 g (99%) colorless product. An analytical sample recrystallized fromCHCl₃ /cyclohexane had mp 69.5°-70.0°; ir (Nujol) 3200, 1760 (broad),1540 cm⁻¹ ; nmr (CDCl₃) 1.35 (t, 3H, J=7), 2.45 (s, 3H), 4.30 (q, 2H,J=7), 5.75 (s, 1H), 9.0 (s, 1H),; and mass spectrum m/e 43 (100%) and207 (M⁺).

Anal. Calcd for C₇ H₁₀ ClNO₄ : C, 40.50; H, 4.85; Cl, 17.08; N, 6.75.Found: C, 40.61; H, 4.61; Cl, 16.92; N, 6.71.

N,N-Dimethyl-2-chloroaceteacetamide

Diketene (42 g, 0.5 mole) was mixed with ice and water (200 ml) andtreated with 25% aq. dimethylamine (90 ml). On completion of theaddition, the solution was allowed to warm to room temperature andstirred for 30 min. It was then cooled with an ice bath, acidified withconc. HCl (250 ml) and treated with aq. sodium hypochlorite ("Chlorox",800 ml) by rapid, dropwise addition. After warming to room temp., theresulting mixture was extracted three times with 400 ml portions ofmethylene chloride. After drying and evaporation of solvent the productwas vacuum distilled to give 67.65 g (83%) of pale yellow liquid with bp97°-8°/0.05 mm; ir film 1730 and 1645 cm⁻¹ ; nmr (CDCl₃) 2.40 (s, 3H),3.03 (s, 3H), 3.20 (s, 3H), and 5.30 ppm (s, 1H); mass spectrum m/e 43and 163 (M⁺).

Anal. Calcd for C₆ H₁₀ ClNO₂ : C, 4405; H, 6.16, Cl, 21.67; N, 8.56.Found: C, 44.31; H, 6.29, Cl, 21.73; N, 8.66.

We claim:
 1. A compound of the structure ##STR5## wherein: R₁ ishydrogen or methylR₂ is chosen from the group consisting of acetyl,propionyl, butyroyl, carbomethoxy, carbethoxy, carbopropionoxy andcarbobutoxy.
 2. A compound of claim 1 wherein R₁ is hydrogen. 3.N-Acetyl-2-chloro-3-oxobutyramide, a compound of claim
 2. 4.N-Carbomethoxy-2-chloro-3-oxobutyramide, a compound of claim
 2. 5.N-Carbethoxy-2-chloro-3-oxobutyramide, a compound of claim 2.